SELENOF Mitigates Bovine Mastitis by Preserving Mitochondrial Homeostasis and Suppressing NLRP3-Mediated Pyroptosis

Bovine mastitis threatens the dairy industry with limited effective therapies. The selenoprotein family offers potential anti-inflammatory interventions, yet the role of Selenoprotein F (SELENOF) remains unclear. This study investigated SELENOF in mitochondrial damage and pyroptosis using clinical mammary biopsies and a <i>Staphylococcus aureus</i>-induced Mammary alveolar cell-type T (MAC-T) cell model. Histology, TEM, immunofluorescence, Western blot, qPCR, RNA-seq, and mitochondrial staining (MitoTracker Red and JC-1) were employed. Mastitic mammary tissue exhibited severe architectural disruption, including focal necrosis with coalescing vacuoles of variable size, extensive epithelial denudation, and interstitial thickening with dense inflammatory infiltrates. At the ultrastructural level, mitochondrial swelling, cristae loss, and plasma membrane rupture were evident. Additionally, these tissue specimens exhibited marked upregulation of inflammatory mediator transcripts, notably <i>IL-1β, IL-6,</i> and <i>TNF-α</i>, alongside heightened abundance of pyroptosis-associated proteins including NOD-like receptor family pyrin domain containing 3 (NLRP3), cleaved caspase-1, and GSDMD-N (Gasdermin D N-terminal domain). RNA-seq identified <i>SELENOF</i> as significantly downregulated. The MAC-T model recapitulated the mitochondrial dysfunction, inflammatory response, and pyroptosis observed in mastitic tissue. <i>SELENOF</i> overexpression restored mitochondrial membrane potential, dampened the output of inflammatory signaling molecules, and suppressed NLRP3-mediated pyroptosis via attenuation of caspase-1/GSDMD-N pathway activation. These findings establish <i>SELENOF</i> as a novel target that mitigates bovine mastitis by preserving mitochondrial homeostasis and suppressing NLRP3-mediated pyroptosis.